Severe lack of functional human tissue and organ-substitutes precludes clinical transplantation for most patients suffering from organ failure. Additionally, robust human models are needed for research and pharmaceutical development. Engineered artificial organs and tissues could provide alternatives or bridges to organ transplant and in vitro systems for drug development or disease modeling. Classical biomaterial- and extracellular matrix-based tissue engineering faces several challenges, such as the unfavorable host response to biomaterials, toxic substances released by scaffold degradation products, and low cellular density. Scaffold- free tissue engineering seeks to create dense 3D tissues only from cells and the matrix they secrete (without the use of polymers and exogenous matrix). To date, such multi-cellular scaffold-free tissues have been created by random mixing of multiple cell types, and any organization has been the result of cellular self- organization1-5. These systems frequently exhibit highly variable cellular morphogenesis (e.g., variable endothelial cell network formation) as well as necrosis at the tissue core. Controlled assembly has been largely unexplored in scaffold-free systems. Indeed, a central unanswered question in tissue engineering is the degree to which engineers will need to control absolute architecture versus coax biology to self-organize into tissues via morphogenesis. We hypothesize that pre-determined spatial organization of cells within engineered tissues will facilitate cellular organization and maximize tissue function, which we define as guided morphogenesis. In this proposal, we will study how spatial signals drive emergent morphogenesis and tissue organization between communities of cells by spatially controlling the initial architecture of engineered scaffold-free multi-cellular tissues. We will develop a platform to micropattern 3D multi-cellular scaffold-free tissues and then study how pre-defined tissue architecture dictates both tissue function and vascular morphogenesis. We will specifically apply these methods to study how modulation of 3D multi-cellular tissue architecture impacts differentiated hepatocyte function and organization as well as microvascular morphogenesis in engineered scaffold-free liver tissue. The ultimate objective of this work is to develop platforms that enable the study of spatially-controlled multicellular interactions in 3D tissue development and function in order to establish architectural design specifications for engineering liver tissue. We expect that such technologies will greatly facilitate the design of future cell-based technologies and engineered tissues. ) PUBLIC HEALTH RELEVANCE: Artificially engineered tissues (e.g., liver) could serve as organ-substitutes for clinical transplantation as well as test systems for research and pharmaceutical development. Here, we will develop novel engineering tools to study how nature organizes different types of cellular building blocks in building three-dimensional tissues. This knowledge could be applied in building functional tissues for human therapies. )